The objective of the proposed research is to unravel the biogenetic pathway used by the mammalian cell to synthesize, assemble, insert, and regulate the concentration of the proteins of the plasma membrane. Toward this objective, we have identified the major polypeptides and glycoproteins forming the plasma membrane of Hepatoma Tissue Culture Cells (HTC cells). Most, but not all, of these polypeptides are externally disposed on the membrane and can be labeled by membrane impermeable probes such as iodination catalysed by lactoperoxidase or borohydride reduction of oxidized (via galactose oxidase) galactose residues. Since these labeling methods do not affect significantly cell metabolism, the fate of the incorporated label can be followed. The polypeptides of the membrane turn over as a unit while the sugar moieties show heterogeneity in their turnover rates. These results imply that membrane proteins are assembled as a unit and we have identified in HTC cells a preformed membrane in an intracellular compartment which is precursor to the plasma membrane. Individual proteins and glycoproteins from both the plasma membrane and the intracellular precursor will be purified, characterized, and compared by tryptic peptide and sugar analysis. Homologous plasma membrane proteins will be purified from rat liver cells and antibodies will be prepared in the expectation that these antibodies will also react with HTC cell proteins. The antibodies then will be used in combination with isotopic methods using amino acid and sugar labels to follow the sequence of synthesis and insertion into the membrane of both the protein backbone and the carbohydrate sidechains of plasma membrane proteins. Finally, attempts will be made to alter the concentration of specific subsets of proteins in the membrane in order to determine whether and how the cell regulates the synthesis, assembly, and insertion of individual proteins in the membrane relative to the other proteins already existing in the preformed membrane pool.